Microbial transformation process for producing antihypertensive N2 -glucuronide products

ABSTRACT

Fermentation of the microorganism Streptomyces sp. MA6751 (ATCC No. 55043) in the presence of the Angiotensin II (A II) receptor antagonist ##STR1## yields an N2-glucuronide analog which is also an A II antagonist useful in the treatment of hypertension and congestive heart failure and other indications known to respond to A II antagonists.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a novel process for the preparation of theantihypertensive agent, compound (I) ##STR2## comprising fermentation ofcompound (II) ##STR3## with the microorganism Streptomyces sp. MA6751(ATCC No. 55043). Compound (I) and its precursor (II) are Angiotensin II(A II) receptor antagonists useful in the treatment of humanhypertensive diseases.

A II is an octapeptide hormone produced mainly in the blood during thecleavage of Angiotensin I by angiotensin converting enzyme (ACE). ACE islocalized on the endothelium of blood vessels of lung, kidney and manyother organs. A II is the end product of the renin-angiotensin system(RAS), a system that plays a central role in the regulation of normalblood pressure and seems to be critically involved in hypertensiondevelopment and maintenance, as well as congestive heart failure. A IIis a powerful arterial vasconstrictor that exerts its action byinteracting with specific receptors present on cell membranes. A IIreceptor antagonism is one of the possible modes of controlling the RAS.

SUMMARY OF THE INVENTION

A new antihypertensive biotransformation product (I) is obtained by thefermentation of the microorganism Streptomyces sp. MA 6751 (ATCC No.55043), in the presence of substrate compound (II). Thebiotransformation is accomplished under submerged aerobic conditions inan aqueous carbohydrate medium containing a nitrogen nutrient at a pH ofabout 7 for a sufficient time to produce compound (I).

The resultant N2-glucuronide analog exhibits A II antagonist activity,i.e., it has an inhibitory concentration (IC₅₀) of less than 50 μm in anassay that measures the concentration of potential A II antagonistneeded to achieve 50% displacement of the total ¹²⁵ I-Sar¹ Ile⁸-angiotensin II specifically bound to a membrane preparation derivedfrom rabbit aortae, and therefore it is an antihypertensive agent. Thisassay is also referred to herein as the "IC₅₀ " assay.

Also provided by this invention is a pharmaceutical compositioncontaining a therapeutically effective amount of compound (I) incombination with a pharmaceutically acceptable non-toxic carrier orexcipient.

In addition, there is provided a method of treating a human host tocontrol hypertensive disorders or for treating congestive heart failurecomprising administering to the host a therapeutically effective amountof compound (I).

DETAILED DESCRIPTION OF THE INVENTION

The product of this invention is compound (I): ##STR4##

The novel process of this invention comprises fermentation of themicroorganism Streptomyces sp. MA6751 in the presence of substratecompound (II) ##STR5## in a nutrient medium, and isolation of theresulting biotransformation product, compound (I), in a conventionalmanner.

A biologically pure sample of Streptomyces sp. MA6751 is currentlyavailable in the permanent culture collection of the American TypeCulture Collection, 12301 Parklawn Drive in Rockville, Md., from whichit is available under the Accession Number ATCC 55043.

The following is a general description of Streptomyces sp. strainMA6751. Observations of growth, general cultural characteristics andcarbon source utilization were made in accordance with the methods ofShirling and Gottleib (Internat. J. System. Bacteriol. 16: 313-340).Chemical composition of the cells was determined using the method ofLechevalier and Lechevalier (in Actinomycete Taxonomy, A. Dietz and D.W. Thayer, Ed. Society for Industrial Microbiology, 1980). Coloration ofthe culture was determined by comparison with color standards containedin the Inter-Society Color Council-National Bureau of Standards CentroidColor Charts (US Dept. of Commerce National Bureau of Standardssupplement to NBS Circular 553, 1985).

Source

The culture was isolated from soil at Heather forest, 9000-13, 400 ft.elevation, Mt. Kilimanjaro, Kenya.

Analysis of Cell Wall Composition

Peptidoglycan contains L-diaminopimelic acid. Whole cell analysis ofcarbohydrates reveals galactose, glucose, mannose and xylose.

General growth characteristics

Good growth on yeast malt extract, (w/o trace elements), glycerolasparagine, inorganic salts-starch, oatmeal, sporulation, and trypticasesoy agars. Growth occurs at 27° and 37° C. Culture also grows well inliquid media such as yeast dextrose broth.

Colony morphology (on yeast malt extract agar)

Substrate mycelium is orange (71 m.OY) and colonies are opaque, raised,lobate and rubbery. The colony surface is rough textured. Aerial myceliaappear after 4 days incubation and are white in color (263 White). Sporemass, when present, is white (263 White).

Micromorphology

Aerial mycelium (0.57-0.76 μm dia.) arises from a substrate mycelium andis branched and flexous. In mature cultures, the aerial myceliumcommonly terminates in spores borne predominantly in spirals and flexouschains.

Miscellaneous physiological reactions

Culture does not produce melanoid pigments in tryptone yeast extractbroth, and yellow, non-pH dependant diffusible pigment is produced onglycerol asparagine agar. Starch is hydrolyzed.

    ______________________________________                                        Cultural Characteristics of Streptomyces sp.                                  MA6751 at 21 Days                                                                                Aerial                                                                        Mycelium                                                             Amount   and/or     Soluble Reverse                                 Medium    Growth   Spores     Pigments                                                                              Color                                   ______________________________________                                        Yeast Extract-                                                                          good     White (263),                                                                             none    Yellow                                  Malt Extract       spiral     noted   (s.Y 87)                                                   sporophores                                                Glucose-  good     White (263),                                                                             Yellow, Yellow                                  Asparagine         spiral     non-pH  (p.Y 89)                                                   sporophores                                                                              dependent                                       Inorganic good     White (263),                                                                             none    Yellow                                  Salts-Starch       spiral     noted   (p.Y 89)                                                   sporophores                                                Oatmeal   good     White (263),                                                                             none    Yellow                                                     spiral     noted   (m.OY 84)                                                  sporophores                                                ______________________________________                                        Carbohydrate Utilization Pattern of Streptomyces sp.                          MA6751 at 21 Days                                                             Carbon source     Utilization*                                                ______________________________________                                        D-arabinose       0                                                           L-arabinose       1                                                           cellobiose        2                                                           D-fructose        2                                                           inositol          2                                                           α-D-lactose 2                                                           β-D-lactose  2                                                           D-maltose         2                                                           D-mannitol        3                                                           D-raffinose       2                                                           L-rhamnose        2                                                           sucrose           2                                                           D-xylose          2                                                           L-xylose          0                                                           α-D-glucose (control)                                                                     3                                                           ______________________________________                                         *3 = good utilization                                                         2 = moderate utilization                                                      1 = poor utilization                                                          0 = no utilization                                                       

In general, compound (I) can be produced by culturing (fermenting) theabove-described microorganism in the presence of an appropriateconcentration of substrate compound (II) in an aqueous nutrient mediumcontaining sources of assimilable carbon and nitrogen, preferably undersubmerged aerobic conditions (e.g. shaking culture, submerged culture,etc.).

Compound (II) may be prepared according to the procedures described inEuropean Patent Application 0253310 A2. An appropriate concentration ofthe parent compound in the aqueous medium ranges from 0.05 mg/ml to 0.2mg/ml, preferably 0.1 mg/ml; less than 0.05 mg/ml is inefficient andgreater than 0.2 mg/ml can inhibit the culture. The aqueous medium isincubated at a temperature between 26° C. and 29° C., preferably 27° C.;culture growth will be inhibited below this temperature range andculture death will occur above this temperature range. The aqueousmedium is incubated for a period of time necessary to complete thebiotransformation as monitored by HPLC, usually for a period of about 4days, on a rotary shaker operating at about 220 rpm. with a throw ofabout 2 in. The aqueous medium is maintained at a pH between 6 and 8,preferably about 7, at the initiation and termination (harvest) of thefermentation process. A higher or lower pH will be detrimental to theviability of the culture. The desired pH may be maintained by the use ofa buffer such as morpholinoethanesulfonic acid (MES),morpholino-propanesulfonic acid (MOPS), and the like, or by choice ofnutrient materials which inherently possess buffering properties, suchas production media described herein below.

The preferred sources of carbon in the nutrient medium are certaincarbohydrates such as glucose, xylose, galactose, glycerin, starch,dextrin, and the like. Other sources which may be included are maltose,rhamnose, raffinose, arabinose, mannose, salicin, sodium succinate, andthe like.

The preferred sources of nitrogen are yeast extract, meat extract,peptone, gluten meal, cottonseed meal, soybean meal and other vegetablemeals (partially or totally defatted), casein hydrolysates, soybeanhydrolysates and yeast hydrolysates, corn steep liquor, dried yeast,wheat germ, feather meal, peanut powder, distiller's solubles, etc., aswell as inorganic and organic nitrogen compounds such as ammonium salts(e.g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.),urea, amino acids, and the like.

The carbon and nitrogen sources, though advantageously employed incombination, need not be used in their pure form because less purematerials which contain traces of growth factors and considerablequantities of mineral nutrients are also suitable for use. When desired,there may be added to the medium mineral salts such as sodium or calciumcarbonate, sodium or potassium phosphate, sodium or potassium chloride,sodium or potassium iodide, magnesium salts, copper salts, cobalt salts,and the like. If necessary, especially when the culture medium foamsseriously, a defoaming agent, such as liquid paraffin, fatty oil, plantoil, mineral oil or silicone may be added.

Submerged aerobic cultural conditions are preferred for the productionof compound (I) in massive amounts. For the production in small amounts,a shaking or surface culture in a flask or bottle is employed.Furthermore, when the growth is carried out in large tanks, it ispreferable to use the vegetative form of the organism for inoculation inthe production tanks in order to avoid growth lag in the process ofproduction of compound (I). Accordingly, it is desirable first toproduce a vegetative inoculum of the organism by inoculating arelatively small quantity of culture medium with spores or mycelia ofthe organism produced in a "slant" and culturing said inoculated medium,also called the "seed medium", and then to transfer the culturedvegetative inoculum aseptically to large tanks.

The fermentation medium, in which the inoculum is produced, issubstantially the same as or different from the medium utilized for theproduction of compound (I) and is generally autoclaved to sterilize themedium prior to inoculation. The fermentation medium is generallyadjusted to a pH between 6 and 8, preferably about 7, prior to theautoclaving step by suitable addition of an acid or base, preferably inthe form of a buffering solution. Temperature of the seed medium ismaintained between 26° C. and 29° C., preferably 27° C.; culture growthwill be inhibited below this range and culture death will occur abovethis range. Incubation of the seed medium is usually conducted for aperiod of about 10 to 30 hours, preferably 24 hours, on a rotary shakeroperating at 220 rpm; the length of incubation time may be variedaccording to fermentation conditions and scales.

Agitation and aeration of the culture mixture may be accomplished in avariety of ways. Agitation may be provided by a propeller or similarmechanical agitation equipment, by revolving or shaking the fermentor,by various pumping equipment or by the passage of sterile air throughthe medium. Aeration may be effected by passing sterile air through thefermentation mixture.

Preferred culturing/production media for carrying out the fermentationinclude the following media:

    ______________________________________                                                          g/l                                                         ______________________________________                                        Seed Medium A                                                                 Dextrose            1.0                                                       Dextrin             10.0                                                      Beef Extract        3.0                                                       Ardamine pH         5.0                                                       NZ Amine Type E     5.0                                                       MgSO.sub.4.7H.sub.2 O                                                                              0.05                                                     K.sub.2 HPO.sub.4    0.37                                                     Adjust pH to 7.1                                                              Add CaCO.sub.3 0.5 g/l                                                        Transformation Medium B                                                       Glucose             10                                                        Hycase SF           2                                                         Beef Extract        1                                                         Corn Steep Liquor   3                                                         Adjust pH to 7.0                                                              ______________________________________                                    

The product, compound (I), can be recovered from the culture medium byconventional means which are commonly used for the recovery of otherknown biologically active substances. Compound (I), 45% yield, is foundin the cultured mycelium and filtrate, which are obtained by filteringor centrifuging the cultured broth, and accordingly can be isolated andpurified from the mycelium and the filtrate by a conventional methodsuch as concentration under reduced pressure, lyophilization, extractionwith a conventional solvent, such as methylene chloride and the like, pHadjustment, treatment with a conventional resin (e.g. anion or cationexchange resin, non-ionic adsorption resin, etc.), treatment with aconventional adsorbent (e.g. activated charcoal, silicic acid, silicagel, cellulose, alumina, etc.), crystallization, recrystallization, andthe like. A preferred recovery method is solvent extraction,particularly using methylene chloride. A preferred purification methodinvolves the use of chromatography, especially high performance liquidchromatography (HPLC), using a silica gel column and an eluant mixturecomposed of water and an organic solvent such as methanol, acetonitrileand the like, and a small amount of acid such as acetic acid,trifluoracetic acid, phosphoric acid and the like. A preferred eluant iscomposed of acetonitrile and water containing 0.1% trifluoroacetic acid,and is run through the column in a linear gradient.

The product of this invention, compound (I), exhibits A II antagonistactivity by the IC₅₀ assay, and therefore is useful in treatinghypertension. It is also of value in the management of acute and chroniccongestive heart failure. This compound may also be expected to beuseful in the treatment of secondary hyperaldosteronism, primary andsecondary pulmonary hyperaldosteronism, primary and secondary pulmonaryhypertension, renal failure such as diabetic nephropathy,glomerulonephritis, scleroderma, glomerular sclerosis, proteinuria ofprimary renal disease, end stage renal disease, renal transplanttherapy, and the like, renal vascular hypertension, left ventriculardysfunction, diabetic retinopathy and in the management of vasculardisorders such as migraine, Raynaud's disease, luminal hyperplasia, andto minimize the atherosclerotic process. The product of this inventionis also useful for cognitive function enhancement. The application ofthe compound of this invention for these and similar disorders will beapparent to those skilled in the art.

The compound of this invention is also useful to treat elevatedintraocular pressure and to enhance retinal blood flow and can beadministered to patients in need of such treatment with typicalpharmaceutical formulations such as tablets, capsules, injectables, aswell as topical ocular formulations in the form of solutions, ointments,inserts, gels, and the like. Pharmaceutical formulations prepared totreat intraocular pressure would typically contain about 0.1 to 15% byweight, preferably 0.5% to 2% by weight of the compound of thisinvention.

In the management of hypertension and the clinical conditions notedabove, the compound of this invention may be utilized in compositionssuch as tablets, capsules or elixirs for oral administration,suppositories for rectal administration, sterile solutions orsuspensions for parenteral or intramuscular administration, and thelike. The compounds of this invention can be administered to patients(animals and human) in need of such treatment in dosages that willprovide optimal pharmaceutical efficacy. Although the dose will varyfrom patient to patient depending upon the nature and severity ofdisease, the patient's weight, special diets then being followed by apatient, concurrent medication, and other factors which those skilled inthe art will recognize, the dosage range will generally be about 1 to1000 mg. per patient per day which can be administered in single ormultiple doses. Perferably, the dosage range will be about 2.5 to 250mg. per patient per day; more preferably about 2.5 to 75 mg. per patientper day.

The compound of this invention can also be administered in combinationwith other antihypertensives such as α-methyldopa, and/or diuretics suchas hydrochlorothiazide, and/or angiotensin converting enzyme inhibitorssuch as enalapril, and/or calcium channel blockers such as nifedipine.Typically, the individual daily dosages for these combinations can rangefrom about one-fifth of the minimally recommended clinical dosages tothe maximum recommended levels for the entities when they are givensingly. These dose ranges can be adjusted on a unit basis as necessaryto permit divided daily dosage and, and as noted above, the dose willvary depending on the nature and severity of the disease, weight of thepatient, special diets and other factors. Typically, these combinationscan be formulated into pharmaceutical compositions as discussed below.

About 1 to 100 mg. of compound (I) or a physiologically acceptable saltthereof is compounded with a physiologically acceptable vehicle,carrier, excipient, binder, preservative, stabilizer, flavor, etc., in aunit dosage form as called for by accepted pharmaceutical practice. Theamount of active substance in these compositions or preparations is suchthat a suitable dosage in the range indicated is obtained.

Illustrative of the adjuvants which can be incorporated in tablets,capsules and the like are the following: a binder such as gumtragacanth, acacia, corn starch or gelatin; an excipient such asmicrocrystalline cellulose; a disintegrating agent such as corn starch,pregelatinized starch, alginic acid and the like; a lubricant such asmagnesium stearate; a sweetening agent such as sucrose, lactose orsaccharin; a flavoring agent such as peppermint, oil of wintergreen orcherry. When the dosage unitform is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier such as fattyoil. Various other materials may be present as coatings or to otherwisemodify the physical form of the dosage unit. For instance, tablets maybe coated with shellac, sugar or both. A syrup or elixir may contain theactive compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavoring such as cherry ororange flavor.

Sterile compositions for injection can be formulated according toconventional pharmaceutical practice by dissolving or suspending theactive substance in a vehicle such as water for injection, a naturallyoccuring vegetable oil like sesame oil, coconut oil, peanut oil,cottonseed oil, etc., or a synthetic fatty vehicle like ethyl oleate orthe like. Buffers, preservatives, antioxidants and the like can beincorporated as required.

Receptor binding assay using rabbit aortae membrane preparation

Three frozen rabbit aortae (obtained from Pel-Freeze Biologicals) weresuspended in 5 mM Tris-0.25M Sucrose, pH 7.4 buffer (50 ml) homogenized,and then centrifuged. The mixture was filtered through a cheesecloth andthe supernatant was centrifuged for 30 minutes at 20,000 rpm at 4° C.The pellet thus obtained was resuspended in 30 ml of 50 mM Tris-5 mMMgCl₂ buffer containing 0.2% Bovine Serum Albumin and 0.2 mg/mlBacitration and the suspension was used for 100 assay tubes. Samplestested for screening were done in duplicate. To the membrane preparation(0.25 ml) there was added ¹²⁵ I-Sar¹ -Ile⁸ -angiotensin II [obtainedfrom New England Nuclear] (10 ul; 20,000 cpm) with or without the testsample and the mixture was incubated at 37° C. for 90 minutes. Themixture was then diluted with ice-cold 50 mM Tris-0.9% NaCl, pH 7.4 (4ml) and filtered through a glass fiber filter (GF/B Whatman 2.4"diameter). The filter was soaked in scintillation cocktail (10 ml) andcounted for radioactivity using Packard 2660 Tricarb liquidscintillation counter. The inhibitory concentration (IC₅₀) of potentialAII antagonist which gives 50% displacement of the total specificallybound ¹²⁵ I-Sar¹ Ile⁸ -angiotensin II was presented as a measure of theefficacy of such compounds as AII antagonists.

Using the methodology described above, the compound of this inventionwas evaluated and was found to exhibit an activity of at least IC₅₀ <50μm thereby demonstrating and confirming the utility of the compound ofthis invention as an effective AII antagonist.

The following examples are given for the purpose of illustrating thepresent invention and should not be construed as being limitations onthe scope or spirit of the instant invention.

EXAMPLE 1 Microorganism and Culture Conditions

A frozen vial (2.0 ml) of culture MA 6751 (ATCC No. 55043) was used toinoculate a 250 ml baffled shake flask containing 50 ml of autoclaved(sterilized) seed medium A. The seed flasks were incubated at 27° C. for24 hours on a rotary shaker operating at 220 rpm. A 2.5 ml aliquot ofthe resulting seed medium was used to inoculate a 250 ml non-baffledshake flasks, each flask containing 50 ml of the previously autoclaved(sterilized) transformation medium B.* Substrate compound (II) was addedto the flask as an aqueous solution with pH of 7 at 0 hour to achieve afinal concentration of 0.1 mg/ml in each flask. The content of the shakeflasks were subsequently incubated for 48 hours at 27° C. on a rotaryshaker operating at 220 rpm. The resultant broths were combined forisolation and purification.

Isolation and Purification Procedure for the Broth

The whole broth was maintained at pH 7.0 and centrifuged. The mycelialcake was washed with water, then discarded. The clear filtrate andwashings were pooled and passed through a Spe-ed Octadecyl cartridge(14% carbon load, Applied Separation, Lehigh Valley, PA) under vacuum.The column was washed with 10% aqueous methanol. Column effluent andwash did not contain microbial product when tested with HPLC. Thecatridge was eluted with methanol. The methanol was evaporated todryness under reduced pressure at 30° C. The resulting oil was dissolvedin methanol and subjected to HPLC purification.

HPLC was carried out on Whatman Magnum 20 Partisil 10 ODS-3 Column (C18,22.1 mm ID×25 cm) at room temperature and monitored at 250 nm. Thecolumn was developed at 6 ml/min with linear gradient from 15% to 80%acetonitrile in 0.1% aqueous phosphoric acid over 80 minutes. Themetabolite fractions were pooled, adjusted to pH 3.0 and evaporated toremove acetonitrile. Desalting was carried out using a C-18 Sep-Pak(Waters Associates) and methanol-water elution solvent to yield pureCompound I.

Characterization

Compound (I) of this invention was characterized via NMR spectrometryyielding the following proton NMR spectrum:

¹ H NMR (250 MHz, CD₃ OD/TMS, ppm), 0.86 (3H, t, J=7.3 Hz), 1.3 (2H, m),1.53 (2H, m), 2.6 (2H, m), 3.62 (2H, m,), 4.05 (2H, m), 4.49 (2H, s),5.31 (2H, s), 5.78 (1H, d, 9.1 Hz), 7.0 & 7.13 (4H, AA'XX', 8.0 Hz), 7.5(3H, m), 7.83 (1H, d, 6.6 Hz).

The mass spectral characteristics are as follows: FAB-MS M+1=599.

What is claimed:
 1. A process for the preparation of a compoundrepresented by formula (I) ##STR6## comprising the steps of culturing amicroorganism Streptomyces sp. MA 6751 (ATCC No. 55043) in a nutrientmedium containing assismilable sources of nitrogen and carbon andsubstrate compound (II) ##STR7## under aerobic conditions until asubstantial amount of the compound is produced and isolating thecompound so produced.
 2. The process of claim 1 wherein the temperatureis 26°-29° C.
 3. The process of claim 1 wherein the temperature is 27°C.